Mutual Effects of Single and Combined CFTR Modulators and Bacterial Infection in Cystic Fibrosis

ABSTRACT Cystic fibrosis transmembrane conductance regulator (CFTR) modulators improve clinical outcomes with varied efficacies in patients with CF. However, the mutual effects of CFTR modulators and bacterial adaptation, together with antibiotic regimens, can influence clinical outcomes. We evaluated the effects of ivacaftor (IVA), lumacaftor (LUM), tezacaftor, elexacaftor, and a three-modulator combination of elexacaftor, tezacaftor, and ivacaftor (ETI), alone or combined with antibiotics, on sequential CF isolates. IVA and ETI showed direct antimicrobial activities against Staphylococcus aureus but not against Pseudomonas aeruginosa. Additive effects or synergies were observed between the CFTR modulators and antibiotics against both species, independently of adaptation to the CF lung. IVA and LUM were the most effective in potentiating antibiotic activity against S. aureus, while IVA and ETI enhanced mainly polymyxin activity against P. aeruginosa. Next, we evaluated the effect of P. aeruginosa pneumonia on the pharmacokinetics of IVA in mice. IVA and its metabolites in plasma, lung, and epithelial lining fluid were increased by P. aeruginosa infection. Thus, CFTR modulators can have direct antimicrobial properties and/or enhance antibiotic activity against initial and adapted S. aureus and P. aeruginosa isolates. Furthermore, bacterial infection impacts airway exposure to IVA, potentially affecting its efficacy. Our findings suggest optimizing host- and pathogen-directed therapies to improve efficacy for personalized treatment. IMPORTANCE CFTR modulators have been developed to correct and/or enhance CFTR activity in patients with specific cystic fibrosis (CF) genotypes. However, it is of great importance to identify potential off-targets of these novel therapies to understand how they affect lung physiology in CF. Since bacterial infections are one of the hallmarks of CF lung disease, the effects (if any) of CFTR modulators on bacteria could impact their efficacy. This work highlights a mutual interaction between CFTR modulators and opportunistic bacterial infections; in particular, it shows that (i) CFTR modulators have an antibacterial activity per se and influence antibiotic efficacy, and (ii) bacterial airway infections affect levels of CFTR modulators in the airways. These findings may help optimize host- and pathogen-directed drug regimens to improve the efficacy of personalized treatment.

I hope you are doing well and missed seeing you at NACFC.
Thank you for submitting your manuscript to Microbiology Spectrum.
I have reviewed your submission for Microbiology Spectrum and have only a few suggestions that I would like you to try to incorporate into this manuscript. I don't think these will take more than some careful rewording or rewriting. In particular: Table 1-I could not figure out how the Staphylococcus aureus isolates in this table related to the patients referred to in Table S2. This needs to be made clearer.
Line 146-I would refer back to Table S3 again here.
Line 159 and beyond-I like the idea of testing IVA in the mouse model of pneumonia. However, I wanted a little more rationale in the Results section of the choice of the acute pneumonia model (rather than a chronic model), the selection of Pseudomonas aeruginosa strain PAO1 (you do mention this eventually in the Discussion) and in the amount of IVA given (which you do refer to the Methods). Realize I have no issue with any of this, but I think better justification up front will head off any criticism.
Finally and perhaps most critically, for the IVA distribution, how do you know whether any of the differences you did see with PAO1 in the mice (Figure 1 A and Tables 4-6) are biologically important? Is there any information about how much IVA is needed to see an increase on gating in mice? Or in any system? In other words, there may be an effect due to infection, but does it even make a difference? I am certainly not proposing that you should do this experiment with CFTR knock out mice, but has anyone else added in IVA to these mice to see what doses are needed to see correct gating? Without this type of information, I think you will need to tone down your conclusions and suggest that these findings may only be relevant under the conditions that you tested (not other strains, doses of PAO1, doses of IVA, etc.).
Minor suggestion-In the headers for the various Results sections, the first time you use abbreviations, please write out the whole words and then put the abbreviations in parentheses. You had this in the tables, but nowhere else. Even I had to look back to remind myself of what an ELF was (not a hobbit :). Sincerely,

Joanna
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Response to editor:
Q1. Table 1-I could not figure out how the Staphylococcus aureus isolates in this table related to the patients referred to in Table S2. This needs to be made clearer. Table S2 including the isolate name. Table S3 again here. Supplementary Table S3. Q3. Line 159 and beyond-I like the idea of testing IVA in the mouse model of pneumonia. However, I wanted a little more rationale in the Results section of the choice of the acute pneumonia model (rather than a chronic model), the selection of Pseudomonas aeruginosa strain PAO1 (you do mention this eventually in the Discussion) and in the amount of IVA given (which you do refer to the Methods). Realize I have no issue with any of this, but I think better justification up front will head off any criticism.

R3.
We thank the editor and agree with this suggestion. In agreement, we have amended the manuscript including details in different sections. Line 162 page 6: "A mouse model of acute infection established with P. aeruginosa reference strain PAO1 was used to mimic early treatment in patients with CF." Line 165 page 6: "Mice were administered IVA at a dose approximately reflecting that used in humans as detailed in Material and Methods." In addition, we comment on the usefulness of extending the study to the chronic infection model in the conclusions. Line 310 page 11. "Furthermore, these analyses should be extended to chronic infection mouse models with repeated administrations to determine the impact both of drug accumulation and lung damage on biodistribution." Q4. Finally and perhaps most critically, for the IVA distribution, how do you know whether any of the differences you did see with PAO1 in the mice (Figure 1 A and Tables 4-6) are biologically important? Is there any information about how much IVA is needed to see an increase on gating in mice? Or in any system? In other words, there may be an effect due to infection, but does it even make a difference? I am certainly not proposing that you should do this experiment with CFTR knock out mice, but has anyone else added in IVA to these mice to see what doses are needed to see correct gating? Without this type of information, I think you will need to tone down your conclusions and suggest that these findings may only be relevant under the conditions that you tested (not other strains, doses of PAO1, doses of IVA, etc.).

R4.
Thank you for your comment. We clarify in the introduction the concept that mouse and human CFTR are different. Thus, mouse models limit efficacy evaluation on CFTR but they are still instrumental for PK studies as investigated in our paper. For these reasons, we do not believe that CFTR mutant mice are relevant. Line 99 page 3. "Mouse models are not helpful for efficacy studies, since cross-species comparative studies have highlighted differences between human and mouse CFTR (33). However, they can facilitate PK analysis and provide much-needed knowledge on the interactions between CFTR modulators and CF pathogens avoiding variables of human studies." Regarding the biological importance of IVA biodistribution in relation to infection, we reported on line 277 page 10: "Notably, we observed antimicrobial activity or additive/synergistic effects with CST at IVA concentrations ranging from 1 to 8 µg/ml, which were comparable to or moderately higher than those observed in the murine ELF." These results suggest the biological relevance of our findings. However, we point out the challenge ahead to further translate our data to humans in the conclusions sections.

Q5.
Minor suggestion-In the headers for the various Results sections, the first time you use abbreviations, please write out the whole words and then put the abbreviations in parentheses. You had this in the tables, but nowhere else. Even I had to look back to remind myself of what an ELF was (not a hobbit :).

R5.
We included these corrections.